Fatty acids containing two or more unsaturated bonds are collectively referred to as polyunsaturated fatty acids (PUFAs) and are known to include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. Some of the polyunsaturated fatty acids cannot be synthesized in the animal body, and such polyunsaturated fatty acids need to be ingested through food, as essential fatty acids. The polyunsaturated fatty acids are widely distributed. For example, arachidonic acid is isolated from lipids extracted from suprarenal glands or livers of animals. Polyunsaturated fatty acids, however, are contained in small amounts in animal organs, and the extraction and isolation of polyunsaturated fatty acids from the animal organs alone are insufficient to supply large amounts of polyunsaturated fatty acids. Microbial techniques have, therefore, been developed for obtaining polyunsaturated fatty acids through cultivation of various microorganisms. In particular, microorganisms of the genera Mortierella are known to produce lipids containing polyunsaturated fatty acids such as arachidonic acid.
Other attempts have also been made to produce polyunsaturated fatty acids in plants. Polyunsaturated fatty acids are known to form reserve lipids such as triacylglycerol (also referred to as triglyceride or TG) and accumulate in microorganism cells or plant seeds.
Triacylglycerol as a reserve lipid is produced in the organism as follows. An acyl group is introduced into glycerol-3-phosphate by glycerol-3-phosphate acyltransferase to generate lysophosphatidic acid, into which an acyl group is introduced by lysophosphate acyltransferase to generate phosphatidic acid. The phosphatidic acid is then dephosphorylated by phosphatidic acid phosphatase to generate diacylglycerol. An acyl group is introduced into the diacylglycerol by diacylglycerol acyltransferase to generate triacylglycerol.
In this pathway, phosphatidic acid (hereinafter also referred to as “PA” or 1,2-diacyl-sn-glycerol-3-phosphate) is a precursor of triacylglycerol as well as a biosynthetic precursor of diacyl glycerophospholipid. In cells such as yeast, phosphatidic acid cytidyltransferase acts on PA and cytidine 5′-triphosphate (CTP) to synthesize CDP diacylglycerol (CDP-DG), which is used for biosynthesis of various phospholipids.
As described above, dephosphorylation of PA for biosynthesis of diacylglycerol (hereinafter also referred to as “DG”) is known to be catalyzed by phosphatidic acid phosphatase (E.C. 3.1.3.4, hereinafter also referred to as “PAP”). The PAP is known to be present in all organisms, from bacteria to vertebrates.
Yeast (Saccharomyces cerevisiae), which is a fungus, is known to have two types of PAPs (Non-Patent Documents 1, 2, and 7). One is a Mg2+-dependent PAP (PAP1), and the other is a Mg2+-independent PAP (PAP2). A PAH1 gene is known as a gene encoding PAP1 (Non-Patent Documents 3 to 5). A pah1Δ mutant also shows a PAP1 activity, which suggests the existence of other genes exhibiting the PAP1 activity. In the pah1Δ mutant, the nuclear membrane and the ER membrane are abnormally dilated, and the expression genes which plays a key role in biosynthesis of phospholipids is abnormally enhanced (Non-Patent Document 6).
On the other hand, known genes encoding PAP2 are DPP1 and LPP1 which exhibit most PAP2 activities in yeast. The enzymes encoded by these genes have broad substrate specificity and are known to act also on, for example, diacylglycerol pyrophosphate (DGPP), lysophosphatidic acid, sphingoid base phosphate, and isoprenoid phosphate to dephosphorylate them.
A lipid-producing fungus, Mortierella alpina, is known to have two types of genes, i.e., a MaPAH1.1 and a MaPAH1.2, as Mg2+-dependent PAP1 homologs (Patent Document 1), and a MaPAP1 gene, which is a Mg2+-independent PAP2 homolog (Patent Document 2).